Others have reported that unfolding free energy is greater by 4.2 or 5.2 kcal per mol, respectively for horse cytochrome c than for yeast iso-2-cytochrome c or a modified yeast iso-1-cytochrome c having Cys 102 blocked with iodoacetamide. The latter value is equivalent to 60 per cent of unfolding free energy of horse cytochrome c. However, the molecular origin of this marked stability difference is not well understood. Thus, understanding of the origin of such a stability difference would help fuller understanding protein stabilization and therefore, the folding process. Our previous studies have allowed us to assign four core domains of cytochrome c. A core domain is a substructure containing a hydrophobic core and the surface (shell) which folds and unfolds as a unit. Core domain 1 folds by itself and consists essentially of the right channel structure (the top and back of the heme) and the heme (the orientation of Dickerson). Core domains 2 and 3, respectively are assigned to the substructures on the left (including the heme Fe-Met80-sulfur bond) and right sides of the heme both including the heme. Core domain 4 is assigned to the substructure at the bottom of the heme including the heme. In our model cytochrome c folds in the order of core domain 1 to 3 to 2 to 4 or 1 to 2 to 3 to 4. Previous studies suggest that development of the marked difference in unfolding free energy between horse and iso-1- cytochromes c correlates with assembly of core domains 1, 2 and 3. Previous studies also suggest that free energy of horse cytochrome c decreases in such a late phase of folding without substantial rearrangement of polar and non-polar residues. Thus, to investigate whether the hydrophobic core residues are responsible for the stability difference in question, we bioengineered a chimera cytochrome c by mutating the entire hydrophobic core residues of iso-2 to those corresponding of horse cytochrome c (horse core-iso-2 shell chimera). Surprisingly, the stability of this chimera was found to be much less than that expected for horse cytochrome c. Thus, hydrophobicity alone appears to be insufficient to account for the marked stability difference between horse and yeast iso-2-cytochromes c. Nevertheless, previous studies suggest that residues of the hydrophobic core are at least in part responsible for stabilization of assembly of core domains 1, 2 and 3 and therefore, the stability differences in question. Thus, we investigated whether there would be cooperation between the hydrophobic core and the surface, which would lower free energy more for horse cytochrome c than for iso-2, using a model system. Others have reported that phosphate binds with horse cytochrome c at least at three sites. Others have reported that phosphate stabilizes cytochrome c. Mutant proteins were prepared by replacing the core or surface residues or both of iso-2-cytochrome c with those corresponding of C102A iso-1-cytochrome c. The stability of phosphate binding was determined by thermodynamic analysis of the wild type and mutant proteins in the presence and absence of phosphate. The thermodynamic data were obtained by measuring UVCD as a function of temperature and pH. The data suggests that there is cooperation between the hydrophobic core and the surface, which stabilizes the phosphate binding.